The major barrier to curing HIV-1 infection is a small pool of resting memory CD4+ T cells that harbor a stably integrated but transcriptionally silent or latent form of the viral genome. In the latent state, the virus can persist despite antiretroviral therapy and antiviral immune responses. Current efforts to cure the infection are focused on directly targeting this latent reservoir. Accurate measurement of the size of the latent reservoir n infected individuals is essential for monitoring eradication efforts. The original assay used to detect latently infected cells was a labor intensive quantitative viral outgrowth assay (Q-VOA) in which a single round of maximal T cell activation was used to induce latently infected cells to produce virus. Simpler PCR-based assays detecting proviral DNA have also been used. We recently demonstrated that PCR-based assays give infected cell frequencies that are at least 2 logs higher than frequencies determined by the Q-VOA. We went on to show that this discrepancy reflects that fact that PCR-based methods dramatically overestimate reservoir size by detecting defective, non-inducible proviruses. However, we have also shown that the Q-VOA, previously considered as the gold standard for reservoir assays, may actually underestimate reservoir size because not all replication-competent proviruses are induced after a single round of maximal T cell activation. In this application, we describe the use of both molecular studies and virus culture experiments to develop a simple PCR-based assay that will accurately determine the frequency of cells with inducible proviruses. Using results from our recently completed analysis of non- induced proviruses, we will first develop PCR-based assays that detect intact proviruses but exclude proviruses with the most common types of defects identified in our study (large internal deletions and APOBEC3G-mediated hypermutation). Using repetitive in vitro stimulation, we will then determine what fraction of the intact proviruses are potentially inducible. If this fraction is consistent from patient to patient, then a simple PCR assay may be used to estimate the size of the reservoir of potentially inducible proviruses. We believe that a simple and scalable assay of this kind will be invaluable for HIV-1 eradication efforts.